1. Field of the Invention
This invention relates to mass spectrometers, and in particular to MALDI ion sources and on-line MALDI ion sources for mass spectrometers. This invention also relates to the field of aerosol mass spectrometry.
2. Background of the Invention
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used extensively for the analysis of nonvolatile and thermally labile biomolecules of large molecular weight. MALDI techniques, as described by Karas and Hillenkamp in Anal. Chem. 1988; 60:2299-2301, and as described by Tanaka et al. in Rapid Commun Mass Spectrom 1988; 2:151-153, the entire contents of which are incorporated herein by reference, can detect molecular ions with masses greater than 100,000 Da.
In a typical MALDI configuration, solid samples are placed under vacuum, where a short-pulsed laser is used to ionize analytes into a mass spectrometer which separates ions according to a mass-to-charge ratio. Almost all MALDI-MS work was initially completed with time-of-flight mass analyzers due to their theoretically unlimited mass range and the requirement of a single start event for data acquisition being well matched with the pulsed laser sources utilized in MALDI.
Recently, ion-traps and Fourier transform ion-cyclotron resonance mass spectrometers have been applied to MALDI configurations. The capability to conduct tandem mass spectrometry (i.e., MSn) has been important for obtaining structural information from MALDI ions which often undergo little or no fragmentation during the desorption/ionization process.
MALDI systems currently in use apply either an UV or IR wavelength laser. Chemical matrix materials, which are combined with the analyte, are chosen to have strong absorption coefficients at the selected laser wavelength. Although the details of the MALDI process are still not definitively known, it is generally believed that energizing the matrix by laser adsorption transfers some of the laser energy to the analyte in a controlled fashion, serving to ionize individual sample molecules.
Sample preparation is paramount to producing good spectral reproducibility and quality in MALDI analyses because changes in the degree of analyte incorporation into matrix crystals affect signal suppression. A variety of matrices and sample preparation techniques have been empirically developed to attempt to create uniform samples and eliminate “sweet spots” (i.e., a term of art used to describe areas that have particularly good spectral response). The most common method utilized in MALDI is a dried droplet method whereby a mixed matrix and an analyte are dispensed together onto a MALDI target plate, and allowed to dry and co-crystallize at room temperature. Other combinations of matrix and analyte dispensing, either mixed or separately, have also been used. Regardless of the sample preparation method, poor uniformity in crystallization results in spot-to-spot differences on the target plate resulting in some sample regions being matrix-rich and not having an optimal matrix-to-analyte molar ratio. Furthermore, despite care in sample preparation, MALDI analysis yields little quantitative information about chemical concentrations.
Fenn et al. in Science, 1989; 246:64-71, the entire contents of which are incorporated herein by reference, describe another soft ionization technique termed electrospray ionization (ESI) used to ionize large biomolecules. In contrast to MALDI, ESI provides a continuous and reproducible source of gas phase ions for MS analysis. ESI utilizes a capillary at high electric potential relative to an opposing plate at near ground potential. Analyte solution contained in the capillary is drawn out by the high electric potential acting on ions in the solution, and small droplets are formed which become ionized when a carrier solvent is vaporized. Because ESI produces analyte gas phase ions from solution, complex MALDI sample preparation techniques of spotting and drying are avoided. However, ESI tends to output multiply charged ions which are difficult to interpret, in contrast to MALDI ions which typically produces singly-charged ions. Thus, there is generally a trade-off between the on-line reproducible analysis from ESI which suffers from complicated interpretation and the off-line less reproducible analysis from MALDI which benefits from simplified interpretation.
Automated sampling handling systems for MALDI analyses have been developed. Automated sampling handling systems range from techniques which load sample plates into vacuum load-locks, such as described for example by Vestal et al. U.S. Pat. No. 5,498,545, the entire contents of which are incorporated herein by reference, to more complicated on-line techniques which attempt to introduce samples into vacuum without contamination, while at the same time evaporating solvent and maintaining the mass spectrometer's vacuum. The latter technique being described for example by Murray, KK. in Mass Spectrom. Rev. 1997; 16:283-299 and by Orsnes et al. Chem Soc Rev 2001; 30:104-112, the entire contents of which are incorporated herein by reference.
A continuous flow (CF) probe is one such on-line technique. In CF-MALDI, a liquid matrix containing a sample is continuously delivered to the end of a probe for laser ionization. By adding a frit to the probe, solid matrices could be crystallized and analyzed with minimal memory effects. Enhancement of the CF-MALDI technique are possible by applying light-absorbing material to the buffer or to the solvent before direct laser vaporization and ionization in the mass spectrometer vacuum. CF-MALDI techniques are described by Yeung et al., U.S. Pat. No. 5,917,185, the entire contents of which are incorporated herein by reference. Another technique involves the use of a rotating ball inlet (ROBIN). ROBIN involves continuous deposition and crystallization of analyte solution onto a rotating surface, followed by transfer into vacuum and direct UV MALDI. One rotating design is based on a rotating quartz wheel where a moving sample holder is applied. This design is described by Karger et al. U.S. Pat. No. 6,175,112, the entire contents of which are incorporated herein by reference. As with CF-MALDI, the ROBIN technique requires adequate surface cleaning procedures to ensure regenerated samples are not contaminated with previous analytes. Because of the delay between sample deposition and analysis, ROBIN is considered to be an “in-line” technique. See for example Foret et al., Proteomics. 2002; vol. 2, pp. 360-372, the entire contents of which are incorporated herein by reference.
One general difficulty with the on-line MALDI-MS techniques is that the sample is often laser desorbed/ionized with a solvent. The solvent can result in adduct formation and lower quality spectra. Furthermore, the challenges in maintaining both sensitivity and mass resolution and the complexity of operating at vacuum pressures have greatly reduced the acceptance of any of these techniques.
Indeed, high vacuum conditions pose significant obstacles to the practical implementation of on-line MALDI-MS. By contrast, the capability of conducting MALDI analyses at atmospheric pressure (AP) greatly simplifies instrumentation. AP-MALDI is a technique which permits MALDI at or near atmospheric pressures. See for example, Laiko et al. U.S. Pat. No. 5,965,884, the entire contents of which are incorporated herein by reference. Comparisons between AP-MALDI and vacuum MALDI spectra show many similarities of singly-charged, intact molecular ions. However, AP-MALDI results reveal even less fragmentation than vacuum MALDI. The soft ionization of AP-MALDI is likely due to collisions of ions with surrounding gas, therefore thermalizing ions before fragmentation occurs, ultimately producing spectra with a high signal-to-noise. Analyte-matrix cluster ions can complicate mass spectra. While cluster effects are mostly absent at m/z values below 2000 Da, declustered by adjusting skimmer-nozzle or skimmer-octapole voltages, depending on the mass spectrometer configuration, can be used. Further matrix declustering can be removed by increasing an intake capillary temperature to the mass analyzer, or by increasing the laser energy. In each case, de-clustering can be affected with ion heating techniques prior to mass analysis.
Developments in AP-MALDI have also demonstrated the ability to conduct laser desorption/ionization without the need for an additional chemical matrix by applying IR irradiation to aqueous solutions. See for example Laiko VV et al., J. Am. Soc. Mass Spectrom. 2002; 13:354-361, the entire contents of which are incorporated herein by reference. Because water has a strong absorption for IR wavelength energy, and aqueous samples can be easily maintained in liquid phase at atmospheric pressure as opposed to vacuum conditions, this matrix-free laser desorption/ionization simplifies MALDI-type sample preparation.
Further, AP-MALDI has the potential to be coupled to liquid solutions via an in-line approach that would deposit sample onto target plates that would be fed into the ion source for analysis. Such an approach, however, introduces delay between sample deposition (and therefore the requisite drying) and analysis. Furthermore, target cleaning and potential contamination of the laser target would have to be controlled. Another approach for on-line AP-MALDI has been described by Orsnes et al. in European Patent App No. 00810890.4, the entire contents of which are incorporated herein by reference. In this technique, a solution is fed to the end of a capillary where a laser is used for desorption/ionization. However, this technique along with all the above mentioned techniques still requires a sample substrate (i.e. a collection surface) for the analyte and matrix. The collection surface poses challenges to reproducibility and frequently introduces contamination.
In vacuum aerosol MALDI described by Murray et al., Anal. Chem. 1994; 66:1601-1609, and Mansoori et al, Anal. Chem. 1996; 68:3595-3601, the entire contents of which are incorporated herein by reference, aerosol techniques have been applied to conduct mass analysis on discrete aerosol particles. In vacuum aerosol MALDI, aerosols from mixed matrix and analyte can be generated at microliter/minute flow rates with nebulizers or piezoelectric aerosol generators. See for example, Murray and He, J. Mass Spectrom. 1999; 34:909-914, the entire contents of which are incorporated herein by reference. By this approach, an aerosol passes from atmospheric pressure to vacuum where particles introduced into the vacuum are available for UV MALDI. One difficulty with this approach is inefficient sample transfer due to a large loss of aerosols in the pumping stages of the inlet, thus consuming large amounts of sample without analysis. Results with vacuum aerosol MALDI show poor mass resolution, likely due to elevated pressures in the mass spectrometer's source from the evaporating solvent.
Aerosols have also been used in mass spectrometer ion sources at atmospheric pressure, but non laser-based ionization techniques have been applied such as field desorption ionization or corona discharge ionization. See for example Berggren et al. U.S. patent application Ser. No. 2002,0166,961, the entire contents of which are incorporated herein by reference. Berggren et al describe a droplet ion source in which individual charged droplets are trapped, then field desorbed and ionized, and finally aerodynamically focused using an aerodynamic lens into a mass spectrometer for analysis. Features of the aerodynamic lens are described by Liu et al. in Aerosol Sci. Technol. 1995; 22:314-324, the entire contents of which are incorporated herein by reference. Without using aerodynamic focusing, the results show a poor transmission of ions into the vacuum of the MS. See for example Feng et al. J. Am. Soc. Mass Spectrom. 11, 393-399 (2000), the entire contents of which are incorporated herein by reference. Berggren et al. describe in U.S. patent application Ser. No. 2002,0158,196, the entire contents of which are incorporated herein by reference, a piezoelectric aerosol generator interfaced to a MS ion source where, once again, field desorption ionization was applied to ionize small droplets. Hager et al. in Appl. Spectrosc., 46, 1460-1463 (1992), the entire contents of which are incorporated herein by reference, describe a technique whereby a neutral aerosol was charged using a corona discharge, but ion sensitivities were not as high as conventional ESI.
Other work with aerosol generators and mass spectrometry have focused on improved sample preparation techniques of target surfaces. See for example, Allmaier G., Rapid Commun. Mass Spectrom. 1997; 11:1567-1569; Ericsson et al., Proteomics. 2001; 1: 1072-1081; and Little et al. Anal. Chem. 1997; 69:4540-4546, the entire contents of which are incorporated herein by reference.
In short, prior techniques have suffered from either substrate contamination issues where matrix enhancement effects have been used or have suffered from compromises in mass spectrometer performance in those techniques which have introduced samples in a “substrate-less” technique into the mass spectrometer. The degradation in mass spectrometer performance can be attributed to the high gas loading occurring as solvents carrying the samples evaporate inside the mass spectrometer creating not only gas loading for the vacuum system of the mass spectrometer, but also potential problems with recondensation of the solvent on electronic components in the mass spectrometer.